Nonstructural protein 3 of bluetongue virus assists virus release by recruiting ESCRT-I protein Tsg101

Abstract

The release of Bluetongue virus (BTV) and other members of the Orbivirus genus from infected host cells occurs predominantly by cell lysis, and in some cases, by budding from the plasma membrane. Two nonstructural proteins, NS3 and NS3A, have been implicated in this process. Here we show that both proteins bind to human Tsg101 and its ortholog from Drosophila melanogaster with similar strengths in vitro. This interaction is mediated by a conserved PSAP motif in NS3 and appears to play a role in virus release. The depletion of Tsg101 with small interfering RNA inhibits the release of BTV and African horse sickness virus, a related orbivirus, from HeLa cells up to fivefold and threefold, respectively. Like most other viral proteins which recruit Tsg101, NS3 also harbors a PPXY late-domain motif that allows NS3 to bind NEDD4-like ubiquitin ligases in vitro. However, the late-domain motifs in NS3 do not function as effectively in facilitating the release of mini Gag virus-like particles from 293T cells as the late domains from human immunodeficiency virus type 1, human T-cell leukemia virus, and Ebola virus. A mutagenesis study showed that the arginine residue in the PPRY motif is responsible for the low activity of the NS3 late-domain motifs. Our data suggest that the BTV late-domain motifs either recruit an antagonist that interferes with budding or fail to recruit an agonist which is different from NEDD4.

FIG. 1.

Cartoon of BTV NS3 and late-domain motifs in orbivirus NS3 proteins. (A) Cartoon showing the domain structure of NS3 and the location and sequence of the late-domain motifs. Transmembrane domains are indicated by dark gray boxes, and the extracellular domain is shown in light gray. The N- and C-terminal intracellular domains extend from amino acids 1 to 117 and 183 to 229, respectively. Hexagons indicate the single glycosylation site, which is encoded by all BTV strains, and “C” indicates the two conserved cysteine residues. The late-domain motifs extend from amino acids 37 to 45. NS3A is generated by initiation at an in-frame ATG at codon 14 of the NS3 open reading frame. (B) Alignment of late-domain motifs in orbivirus NS3 proteins. Numbers to the right indicate the amino acid positions of the aligned sequences within NS3.

FIG. 2.

Pull-down analysis of the interaction between NS3 and Tsg101. (A) NS3 of BTV-10 was expressed in Sf21 cells, using a recombinant baculovirus, and incubated with GST-agarose beads (lane 1), GST-tagged p11 (lanes 3 and 4), and GST-tagged UEV of human Tsg101 (lanes 6 and 7). Two different buffers (see Materials and Methods) were used for solubilizing NS3 during incubation and for washing of GST-p11 and GST-UEV protein complexes, which was followed by immunoblotting for NS3. (B) NS3 of BTV-10 was expressed in 293T cells and used in pull-down assays with GST-tagged UEV or GST-tagged Alix lacking the C-terminal PRD domain. A whole-cell lysate corresponding to 1% of the amount used for pull-down assays was run in lane 1 for comparison. Note that the transient expression of BTV-10 NS3 in mammalian cells gives rise to two bands, which are formed at approximately equal levels and represent the glycosylated (GNS3) and nonglycosylated (NS3) protein. (C) HIV-1 Gag was expressed in 293T cells, mixed with NS3A of BTV-10 or NS3 of AHSV-6, and used for pull-down assays with GST-tagged UEV of human Tsg101 (hTsg) or its orthologue from Drosophila melanogaster (dTsg). The amount of GST-tagged protein was estimated to be about 10 μg in lanes 1, 3, 5, and 7 and 2 μg in lanes 2, 4, 6, and 8. Western blotting was performed with a mixture of polyclonal antiserum against NS3 and a monoclonal antibody against p24 of HIV Gag.

FIG. 3.

Orbivirus NS3 binds to Tsg101 by means of PSAP or ASAP motifs. (A) Mutations introduced into BTV-10 NS3 for analyzing binding specificity. Five mutants were generated by introducing single or double mutations into NS3 and NS3A. The substitutions are enclosed by rectangles and are aligned with the wild-type sequence of codons 35 to 55 of NS3. (B) Pull-down analysis of the interaction between NS3 and Tsg101. Lysates of 293T cells expressing NS3, NS3A, or one of three different mutants were incubated with GST-UEV (from human Tsg101) or GST-p11, followed by immunoblot analysis for NS3. (C) Pull-down analysis of the interaction between NS3A and Tsg101 (left panel). Lysates of 293T cells expressing NS3A or one of four different mutants were incubated with GST-UEV (from human Tsg101), followed by immunoblot analysis for NS3A. The right panel shows the amounts of input proteins for polyacrylamide gel electrophoresis.

FIG. 4.

Confocal images of HeLa cells showing colocalization of NS3 and GFP-tagged Tsg101. Expression plasmids for NS3 and GFP-tagged Tsg101 were cotransfected into HeLa cells. At 24 h posttransfection, the cells were fixed and processed for confocal microscopy. NS3 (red) was detected by indirect immunofluorescence.

FIG. 5.

NS3 binds to NEDD4-like ubiquitin ligases in vitro. Lysates of 293T cells expressing NS3A or one of three different mutants were incubated with GST-tagged WW domains of the human ubiquitin ligases WWP1, Itch, and NEDD4.1, followed by immunoblot analysis of bound NS3. The mutants are shown in more detail in Fig. 3.

FIG. 6.

Depletion of Tsg101 inhibits release of BTV-10. HeLa cells were transfected twice with the indicated siRNAs and infected 24 h later with BTV-10. Cell lysates were harvested at 24 h postinfection and analyzed by immunoblotting with antisera specific for Tsg101, NS3, and the heavy chain of annexin II (p36) (A) or with polyclonal antisera against BTV-10 and tubulin (B). (C) Virus titers in the supernatant were determined at 12 h postinfection and plotted as percentages of the titer in untransfected cells. Bars show the means of three independent experiments. (D) The ratio of extracellular to intracellular virus was determined after the cells were lysed and was plotted as a percentage of the ratio of untransfected cells. (E) Logarithmic values of intracellular virus titers of transfected and untransfected cells.

FIG. 7.

Depletion of Tsg101 with siRNA inhibits release of BTV-2 and AHSV-6. HeLa cells were transfected twice with the indicated siRNAs and infected 24 h later with BTV-2 (A) or AHSV-6 (B). Two siRNAs against Tsg101 were used (Tsg si1 [striped bars] and Tsg si2 [gray bars]). Virus titers were determined at 12 and 24 h posttransfection and plotted as percentages of the titer in untransfected cells. For negative controls, p36 siRNA (p36 control) (white bars) and a 21-nucleotide antisense RNA (Tsg control) (black bars) against a target sequence within Tsg101 were used.

FIG. 8.

Schematic illustration of constructs used in VLP release assays. Wild-type HIV-1 Gag is represented at the top by rectangular boxes, and individual Gag proteins are indicated inside the boxes. Vertical lines represent the boundaries between the mature cleavage products. Deletions are indicated by thin lines, and numbers are used to indicate the amino acid position and size of each deletion. Wavy lines represent the myristyl group at the N terminus of Gag. H6 was derived from wild-type Gag by deleting amino acids 10 to 278 and was used as a positive control. H6ko was used as a negative control, as it lacks the C-terminal 14 amino acids of p6 and also carries three substitutions in the PTAP motif, which render the construct defective in the late domain. The construct also carries a C-terminal extension of 18 amino acids derived from vector sequences, which were replaced with oligonucleotides or PCR products to generate the other constructs shown in the cartoons. Codons 33 to 49 of BTV-10 NS3 were appended to p6 to generate BTLD, codons 5 to 16 of Ebola virus VP40 were added to generate EBLD, and codons 117 to 129 of HTLV-1 Gag were added to generate HTLD. PCR products encompassing codons 1 to 117 of wild-type or mutant NS3 were added to generate constructs NS3, M1, and M2. WT, wild type.

FIG. 9.

Release of virus-like particles from cells expressing HIV Gag constructs. 293T cells were transfected with the constructs shown in Fig. 8. Virus-like particles were harvested from the supernatant at 24 h posttransfection and analyzed by immunoblotting with an antibody against p24. (A) The NS3 N-terminal domain induces particle release when fused to a late-domain-defective HIV Gag mutant. (B) The NS3 late-domain (BTLD) motifs induce particle release but are less active than the late-domain motifs of Ebola virus (EBLD) and HTLV (HTLD). The upper panel shows particles released into the medium. The lower panel shows whole-cell lysates for comparison to confirm that the proteins were expressed at approximately equal levels. (C) The arginine at position three of the PPRY motif is mainly responsible for the low activity of BTV NS3 in promoting particle release from mammalian cells. Mutated amino acids are underlined in each construct. The first lane shows the negative control (a late-domain-defective Gag mutant). The last lane (lane 12) shows the positive control (a Gag mutant carrying wild-type p6). Note the decrease in electrophoretic mobility when arginine is replaced by proline, which is particularly pronounced in the HTLV background. The construct shown in lane 2 carries amino acids 23 to 38 of AHSV-3 NS3.

FIG. 10.

Release of virus-like particles from cells expressing HIV Gag constructs. (A) Schematic representation of the HIV Gag construct H6Tko, which carries a knockout mutation in the PTAP motif, and the H6Ako construct, which lacks the binding site for Alix. The late-domain motifs of BTV NS3 and Ebola virus VP40 were appended to the C termini of both constructs, as indicated in the lower panel. Numbers indicate amino acid positions. (B) Gag fusion constructs and parent plasmids were transfected into 293T cells, and VLP release was monitored as described in the legend to Fig. 8. (C) Whole-cell lysates of transfected cells were analyzed with Gag antiserum to verify that the constructs were expressed at equal levels.